The Regulation Of Glucose And Lipid Metabolism On Food Intake Of Grass Carp And Mandarin Fish

Feeding is the fundamental behavior of fish.Food intake is a complex behavior which influenced by external factors(such as the environment,season,food supply,stress)and internal factors(sensory system,nutrients,circulating hormone levels).It is known that fish feeding is jointly regulated by central nervous system and peripheral satiety system and fish appetite is regulated by orexigenic and anorexigenic factors.However,the intricate mechanism involved in fish appetite regulation still waits for further investigation.To elucidate the molecular mechanism of grass carp(Ctenopharyngodon idella)and mandarin fish(Siniperca chuatsi)feeding regulation,using modern biological technology such as molecular cloning,gene regulation,heterologous expression,we study the differential responses of sweet taste receptors on glucose or fructose,in vivo administration of leptin or ghrelin on food intake and appetite,and the feedback regulation of dietary carbohydrate or lipid on feeding.In the first part,to explore the relationship between taste receptors and food stimulus,we analyzed the gene structure and evolutionary characteristics of sweet receptors of grass carp and their differential signal transduction elicited by glucose and fructose.Here,we identified six taste receptor genes(gcT1R2s)in typical herbivorous fish,grass carp.Evolutionary analyses showed that the new formed four gcT1R2s(gcT1R2C,gcT1R2D,gcT1R2E,gcT1R2F)were formed as paralogous genes after the formation of gcT1R2A.All six gcT1R2s were prominently expressed in taste organs and their corresponding proteins mediated glucose-or fructose-induced production of intracellular calcium in vitro,indicating they were all functional genes.Particularly,co-expressed six gcT1R2s/gcT1R3increased intracellular Ca²⁺levels to both glucose and fructose treatment,whereas co-expression of novel four gcT1R2s/gcT1R3 only elevated the perception of plant specific fructose,raising the possibility that the formation of new gcT1R2 copies in grass carp were related to its dietary switch to herbivory.Our results suggested that the evolution process of taste not only includes pseudogene in mammals and gene loss in birds,but also includes adaptive gene expansion as demonstrated in fish,and even related to vegetarian adaptation.In the second part,to reveal the feeding regulation of peripheral key appetite control factor in fish,we monitored the change of food intake and appetite after in vivo administration of leptin or ghrelin in grass carp or mandarin fish.The well-known satiety signal factor,leptin,is secreted by the liver in fish.For the first time,the fish leptin A and leptin B were expressed successfully using in vitro recombinant expression protein technology.Taking advantage of this,we further explored the physiological effects of leptin A and leptin B in mandarin fish through intracerebroventricular and intraperitoneal(IP)administration.Results showed that the leptin B was more powerful than leptin A in inhibiting food intake by downregulating the expression of orexigenic genes(NPY/Ag RP)in a short term.Moreover,an adverse feedback regulation of hepatic glycogen with long-term intraperitoneal leptin B injection were observed,whereas the leptin A might play a role in hepatic lipid regulation.Two gastrointestinal peptides ghrelin and obestatin derived from post-translational processing of a common precursor preproghrelin.Fish received IP of saline,ghrelin(100ng/g BW),obestatin-like(25 ng/g BW)and ghrelin in combination with obestatin-like.Ghrelin stimulation of food intake varied considerably among individual fish with 70.8%eliciting a robust response.In these high-responders,food intake was significantly increased by IP ghrelin within 2h.IP obestatin-like alone failed to change food intake in grass carp.However,co-administration of ghrelin and obestatin-like resulted in a decrease in food intake,indicating that obestatin-like peptide partially blocked the ghrelin-induced appetite,suggesting the possible involvement of obestatin as a mediator of the ghrelin stimulatory action on food intake,at least in“high-responders”in grass carp.In addition,we obtained the full-length preproghrelin c DNA in Chinese perch.The tissue distribution of preproghrelin m RNA showed that it was ubiquitously expressed and had significantly higher expression levels in stomach and pituitary than other tissues.The fasting study showed,after gastric emptying(3-6 h),short-term fasting(6-12 h)increased preproghrelin expression in the stomach.While in the pituitary,fasting reduced preproghrelin expression at 1,3,12,and 48 h,suggesting the presence of state fluctuation of self-adjustment.Six-hour-fasting treatment after gastric emptying led to higher preproghrelin expression,indicated potential appetite increase in Chinese perch.In the third part,to study the feedback regulation of dietary carbohydrate or lipid on feeding,grass carp were fed with different dietary carbohydrate and fat levels for 8 weeks.After 8-week feeding trial,the maximal growth performance and feed utilization of grass carp were observed when the dietary carbohydrate level were 218.1 g/kg and the dietary fat level were 43.5 g/kg.The metabolic adaptation strategy of energy changes had been analysed through the serum biochemical components and key gene expressions of glucose-and lipid-metabolism in grass carp fed with different dietary ingredients;On the other hand,the results of the food intake and appetite gene expressions,and the expression of leptin showed that the increase in glucose and fatty acids might induce the appetite suppression by high dietary glucose or lipid inatke through modulation of leptin expression in grass carp.Based on the studies of the relationship between taste receptors and feeding,the feeding regulation of peripheral key appetite factors,and the feedback regulation of dietary glucose or lipid,our findings could greatly enrich the information of fish feeding regulation mechanism,then promote the feed efficiency and decrease the water pollution.